Asian Journal of Agricultural Sciences 5(2): 25-31, 2013
ISSN: 2041-3882; e-ISSN: 2041-3890
© Maxwell Scientific Organization, 2013
Submitted: December 10, 2012
Accepted: January 17, 2013
Published: March 25, 2013
Genetic Diversity in Certain Genotypes of Chilli and Paprika as Revealed
by RAPD and SSR Analysis
1
1
Samuel Tilahun, 2P. Paramaguru and 3J.R. Kannan Bapu
Ethiopian Institute of Agricultural Research, Holetta Agricultural Research
Center, P.O. Box 25766, Addis Ababa, Ethiopia
2
Horticultural College and Research Institute,
3
Center for Plant Breeding and Genetics, Tamil Nadu Agricultural
University, Coimbatore 641-003, India
Abstract: Genetic relationships between thirteen genotypes of chilli and paprika collected from different places of
India were analyzed using twelve RAPD and nine SSR primers. Clustering based on the generated markers was
conducted using NTSYS software’s. The RAPD primers produced a total of 78 bands of which 65 were
polymorphic with a mean of 5.41 polymorphic bands per primer while the SSR primers produced 28 SSR loci with a
mean of 3.22 alleles per SSR locus. The similarity index matrix ranged from 0.36 to 0.91 (RAPD) and 0.117 to 0.9
(SSR) with mean of 0.62 and 0.39 respectively. Polymorphic Information Content (PIC) values of the SSR primers
ranged from 0.00 to 0.75 with an average value of 0.546. The resulting RAPD and SSR dendrograms separated the
cultivars with small and erect fruit position from the large and medium fruited cultivars with declining fruit position.
Both RAPD and SSR markers showed genetic variability in the studied pepper genotypes and they are powerful
tools for estimating genetic similarities and diversity.
Keywords: Dendrogram, polymorphism, randomly amplified polymorphic DNA, simple sequence repeats
Minamiyama et al., 2006; Portis et al., 2007; Hanáček
et al., 2009; Parmar et al., 2010).
RAPD (Randomly Amplified Polymorphic DNA)
markers are not affected by the prevailing
environmental conditions and present in all plant. They
can identify a great number of polymorphisms that
allow the distinction among accessions and the
identification of possible duplicates (Ilbi, 2003).
RAPDs are considered advantageous to analyze the
genetic diversity of germplasms for the reason that they
are simple, cheap and give quick results (Costa et al.,
2006). But they only identify dominant alleles and have
reproducibility problem.
Recently, microsatellite markers have been applied
to study phylogeny and construction of genetic maps
in pepper. These microsatellites (Simple Sequence
Repeats, SSRs) are having particular significance
because of their co-dominance, which is lacking in
RAPD markers and multiple allelic nature. Moreover,
SSRs are abundant and hyper variable in almost all
genomes examined to date (Powell et al., 1996). A
number of SSRs markers have been developed from
genomic DNA, ESTs (Expression Sequence Tags) and
cDNAs which can be used for the diversity analysis of
pepper genetic resources (Minamiyama et al., 2006;
Portis et al., 2007).
INTRODUCTION
The Capsicum genus originated from tropical and
humid zone of Central and Southern America and
belongs to the Solanaceae family. Among the
domesticated Capsicum species, pungent and nonpungent forms of Capsicum annuum L. (pepper) are
most popular and have a worldwide commercial
distribution (Bosland and Votava, 2000). In India, hot
pepper or chilli is an important commercial crop which
is cultivated for vegetable, spice and value-added
processed product. A wide range of variability is
reported in the crop (Sreelathakumary and Rajamony,
2004).
Subjective classification based on morphological
data has been reported to create confusion and difficulty
in classifying the genus capsicum (Costa et al., 2006).
In addition, the level of polymorphism for
morphological characteristics in genotypes is
sometimes so limited that it is inadequate to allow
varietal identification (Ilbi, 2003). In the past several
years a range of molecular markers such as RFLPs,
AFLP, RAPDs and SSRs have been used for the
estimation of genetic diversity, genome mapping and
gene tagging in the capsicum species and related crops.
(Lefebvre et al., 1995; Paran et al., 1998; Rodriguez
et al., 1999; Thorup et al., 2000; Lefebvre et al., 2001;
Corresponding Author: Samuel Tilahun, Ethiopian Institute of Agricultural Research, Holetta Agricultural Research Center,
P.O. Box 25766, Addis Ababa, Ethiopia
25
Asian J. Agric. Sci., 5(2): 25-31, 2013
Table 1: Details of chilli and paprika genotypes used in this study
Genotypes
Geographical origin
Chilli type
CA 97
Tamil Nadu, India
CCH
Tamil Nadu, India
CF 1
Tamil Nadu, India
CO 2
Tamil Nadu, India
K1
Tamil Nadu, India
CA 110
Tamil Nadu, India
Kashi anmol
Uttar Pradesh, India
Paprika type
Bayadagi-kaddi
Karnataka, India
KT-PL-19
Himachal Pradesh, India
KT-PL 18
Himachal Pradesh, India
ARKA ABHIR
Karnataka, India
VSH
V1
Characteristic
Medium fruit, erect fruit position
Medium fruit, up right growth
Small fruit, erect fruit position
Bold fruit, prostate growth
Large fruit, up right growth
Medium fruit, prostate growth, yellow anther
Medium fruit, prostate growth
Large fruit, up right growth
Large fruit, up right growth
Large fruit, up right growth
Large fruit, up right growth
Large fruit, up right growth, orange fruit
Medium fruit, up right growth, yellow fruit
Table 2: Primers sequences with their polymorphic percentage obtained from the twelve RAPD markers
Primer code (RAPD)
Sequence (5´-3´)
Total bands
Polymorphic bands
OPA 1
CAGGCCCTTC
8
8
OPA 2
TGCCGAGCTG
6
3
OPB 3
CATCCCCCTG
5
4
OPB 6
TGCTCTGCCC
4
4
OPF 9
CCAAGCTTCC
5
5
OPF 11
TTGGTACCCC
9
6
OPF 14
TGCTGCAGGT
5
5
OPF 15
CCAGTACTCC
6
6
OPF 16
GGAGTACTGG
9
7
OPF 18
TTCCCGGGTT
10
8
OPF 19
CCTCTAGACC
5
5
OPF 20
GGTCTAGAGG
6
4
The basis and the pattern of variations that are
presented in genetic resources have considerable role in
the success of plant breeding programs. Therefore, this
investigation was undertaken with the objective of
estimating the genetic diversity presented in chilli and
paprika accessions/varieties using RAPD and SSR
markers.
Polymorphic percentage (%)
100
50
80
100
100
66.66
100
100
77.77
80
100
66.66
chloroform: isoamyl alcohol (24:1) and centrifuged at
10,000 rpm for 10 min. This step was repeated once
more and a volume of cold isopropanol was added to
the aqueous phase and centrifuged at 10,000 rpm for
10 min.
The collected precipitate was washed with 20 mL
of 70% ethanol and air dried. After drying the pellet
was dissolved in 3 mL double-distilled water and
treated with RNaseA (15 µL) at 37°C for 30 min. Then,
5 M sodium chloride (1/10 volumes) and cold ethanol
(3 volumes), were added to the solution and centrifuged
at 10,000 rpm for 20 min. Finally, after washing the
recovered pellets with 20 mL of 70% ethanol, the DNA
was dissolved in double distilled water. Integrity and
size of genomic DNA was evaluated on Gel
electrophoresis.
MATERIALS AND METHODS
Plant materials: Seeds of thirteen varieties and
accessions of chilli and paprika collected from different
places of India were obtained from Horticultural
College and Research Institute, Tamil Nadu
Agricultural University, India (Table 1). The genotypes
were grown in pots placed in a 35% shade net under the
regular agronomic practices.
RAPD analysis: The thirteen DNA samples were
analyzed by 12 arbitrary oligodeoxynucleotide primers
(10-mer) selected from those that had reproducible
amplification patterns in RAPD PCR test (Table 2). The
optimized PCR assay was done in a 25 µL reaction
volume containing 2.5 mM MgCl2, 200 µM of dNTP
mix, 20 pmoL of single RAPD primer, 50 ng of pepper
genomic DNA as template and 0.3 U Taq DNA
polymerase in PCR reaction buffer (Genei, Bangalore,
India).
BIORAD (BioRad Laboratories, Inc. USA) with
heated lid was programmed with an initial incubation at
94°C for 4 min followed by 45 amplification cycles
DNA extraction: Healthy and fully expanded leaves
(1 g) were collected from four or five plant for each
accession. Bulked samples were powdered using mortar
and pestle in presence of liquid nitrogen. DNA
extractions were performed according to the procedure
of De Masi et al. (2006) with some modification.
In brief, the powder was suspended in 10 mL of
CTAB lysis buffer consisting of 1.4 M NaCl, 2% w/v
Cetyltrimethyl Ammonium Bromide (CTAB), 100 mM
Tris HCl pH 8.0, 20 mM EDTA, 1% PVP, 1.4% v/v 2mercaptoethanol and incubated at 65oC for 30 min. The
samples were then treated by equal volume of
26 Asian J. Agric. Sci., 5(2): 25-31, 2013
made of three steps: denaturation at 94°C for 1 min,
annealing at 36°C for 1 min and elongation at 72°C for
1½ min. The cycles were ended with final elongation at
72°C for 10 min.
SSR analysis: Nine SSR markers described previously
for pepper were used (Lee et al., 2004; Minamiyama
et al., 2006). The 25 μL-reaction mixture for PCR
contained: 50 ng template DNA, 1 U Taq polymerase
0.2 µM of forward primer, 0.2 µM of reverse primer
and 0.2 mM dNTPs all in PCR reaction buffer (Genei,
Bangalore India).
PCR amplification was done in the BIORAD
thermal cycler (Bio-Rad Laboratories, Inc. USA) set
with initial denaturation step at 94°C for 3 min
followed by 35 cycles of 94°C for 1 min, 55°C for
1minute and 72°C for 2 min. The reaction was
terminated by a final extension step at 72°C for 10 min.
Fig. 1: PCR amplification pattern obtained from thirteen
capsicum genotypes using RAPD primer OPF-20
Lane 1: CA97; 2: CCH; 3: CF1; 4: CO2; 5: K1; 6:
CA110; 7: Kashi anmol; 8: Arka abhir; 9: ktpl 19; 10:
ktpl 18; 11: Byadagikaddi; 12: V1; 13: VSH; M:
Molecular size marker (100 bp ladder, Gene link)
Gel electrophoresis: The RAPD and SSR amplicons
were separated by 1X TBE buffered electrophoresis on
2% w/v agarose gel, containing ethidium bromide (0.5
μg/mL). RAPD amplicons were run for 2 h at 70 V,
while the SSR amplicons were run at 120 V for 1½ h.
The resultant bands were visualized under UV light and
digitized by imaging System (G: BOX, SYNGENE,
UK). Size of amplicons was estimated using a 100 bp
DNA ladder as molecular weight standard (Genei,
Bangalore).
Data analysis: Reproducible and bright RAPD and
SSR fragments were scored in each genotype as “1” if
present or as “0” if absent. The matrices were entered
into the NTSYS program (Numerical Taxonomy
System, version 2.02, Rohlf, 1998) and used to calculate
the pair wise similarity among the genotypes according
to Jaccard’s similarity coefficient:
Fig. 2: Dendrogram of thirteen pepper accessions and
varieties using UPGMA cluster analysis
PIC = 1-∑
2
i=1
where, f2 is the frequency of the ith allele.
RESULTS
Sij
The 12 RAPD primers used in the study amplified
a total of 78 bands and 65 polymorphic bands (Table 2).
The number of polymorphic bands for each primer
varied from 3 (OPB 6) to 8 (OPA 1, OPF 18) with an
average of 5.41 bands per primer. The primers OPA 1,
OPB6, OPF 9, OPF14 OPF15 and OPF19 were the
most polymorphic among all primers tested, generating
100% polymorphism while the primers (OPA 2)
showed the lowest 50% polymorphism. The distribution
pattern of RAPD markers across the thirteen pepper
varieties is shown in (Fig. 1).
The similarity coefficient ranged from 0.36 to 0.91
with a mean value of 0.60. Pair wise similarity between
(Byadagi Kaddi and CF1) and (CA97 and CF1) showed
the highest genetic diversity with the value of 0.36 and
0.41 similarity coefficients, respectively (Table 3).
Whereas, the highest similarity value of 0.91 was found
between KT PL 19 and KT PL 18. The dendrogram
generated based on RAPD data resulted in four major
clusters (Fig. 2). One genotype (CA 97) with
where
Sij : The measurement of the genetic similarity between
individuals i and j
a : The number of polymorphic bands present in both
individuals
bi : The number of bands present in i and absent in j
cj : The number of bands present in j and absent in i
The Similarity coefficients were used to construct a
dendrogram using the UPGMA (unweighted pair group
method with arithmetic average) and the SHAN
(sequential, hierarchical and nested clustering)
procedures in the NTSYS program.
Polymorphic information content: Polymorphism
Information Content (PIC) value was estimated using
the equation given by Smith et al. (1997):
27 Asian J. Agric. Sci., 5(2): 25-31, 2013
Table 3: Similarity matrix of chillies and paprika generated by jaccard’s similarity index based on RAPD and SSR markers
CA 97 CCH
CF 1
CO 2
K1
CA 110 Kashi
Arka
KT-PL- KT-PL- Bayadagi
Anmol
Abhir
19
18
Kaddi
CA 97
1.00
CCH
0.621
1.00
(0.22)2
CF 1
0.41
0.49
1.00
(0.31)
(0.31)
CO 2
0.51
0.64
0.43
1.00
(0.43)
(0.4)
(0.31)
K1
0.56
0.84
0.51
0.70
1.00
(0.36)
(0.54)
(0.17)
(0.58)
CA 110
0.57
0.68
0.45
0.74
0.77
1.00
(0.36)
(0.54)
(0.17)
(0.36)
(0.38)
Kashi Anmol 0.56
0.62
0.42
0.57
0.67
0.62
1.00
(0.44)
(0.44)
(0.50)
(0.26)
(0.44)
(0.57)
Arkaabhir
0.56
0.67
0.44
0.57
0.70
0.64
0.74
1.00
(0.29)
(0.44)
(0.21)
(0.29)
(0.50)
(0.50)
(0.66)
KT-PL-19
0.51
0.72
0.48
0.69
0.86
0.72
0.66
0.69
1.00
(0.46)
(0.54)
(0.17)
(0.46)
(0.50)
(0.50)
(0.57)
(0.40)
KT-PL-18
0.47
0.68
0.44
0.74
0.78
0.68
0.61
0.61
0.91
1.00
(0.20)
(0.26)
(0.12)
(0.20)
(0.21)
(0.21)
(0.31)
(0.25)
(0.55)
Bayadagi
0.49
0.57
0.36
0.52
0.56
0.60
0.63
0.66
0.55
0.50
1.00
Kaddi
(0.28)
(0.46)
(0.19)
(0.20)
(0.31)
(0.55)
(0.40)
(0.54)
(0.42)
(0.23)
V-1
0.57
0.62
0.46
0.61
0.67
0.68
0.72
0.78
0.66
0.62
0.67
(0.33)
(0.61)
(0.23)
(0.43)
(0.46)
(0.90)
(0.53)
(0.57)
(0.46)
(0.20)
(0.50)
VSH
0.63
0.69
0.48
0.59
0.65
0.63
0.73
0.82
0.64
0.60
0.68
(0.5)
(0.46)
(0.29)
(0.50)
(0.43)
(0.33)
(0.5)
(0.43)
(0.54)
(0.46)
(0.36)
1
: Similarity coefficients obtained from RAPD markers; 2: Similarity coefficients obtained from SSR markers
V-1
VSH
1.00
0.83
1.00
(0.40)
Table 4: Primers sequences with their Polymorphism Information Content (PIC) values from SSR markers examined in the thirteen pepper
varieties/accessions
Primer code (SSR)
Sequence (5´-3´)
Repeat motifs
Number of alleles
PIC
2
0.26
CAMS-117
F: TTGTGGAGGAAACAAGCAAA
R: CCTCAGCCCAGGAGACATAA
(tg)21 (ta)3
CAMS-405
F: TTCTTGGGTCCCACACTTTC
(tc)18
2
0.43
R: AGGTTGAAAGGAGGGCAATA
6
0.75
CAMS-864
F: CTGTTGTGGAAGAAGAGGACA
R: GCTTCTTTTTCAACCTCCTCCT
(aga)32
5
0.66
GPMS 113
F: GCACAAGTCAATCCAAACGA
R: CAAAAAGATGATGATGGATGAGA
(at)20 (ag)18
5
0.72
GPMS 161
F: CGAAATCCAATAAACGAGTGAAG
R: CCTGTGTGAACAAGTTTTCAGG
(aat)25
3
0.53
GPMS 197
F: GCAGAGAAAATAAAATTCTCGG
R: CAATGGAAATTTCATCGACG
(ga)3 (tat)16
2
0.48
EPMS 303
F: AAAACTCCAAACTACCCCTGG
R: TTAAGCGTAGCGCTTGTGTC
(ta)25
1
0
EPMS 418
F: ATCTTCTTCTCATTTCTCCCTTC
R: TGCTCAGCATTAACGACGTC
(ca)10
3
0.40
CAMS-327
F: GCATCTAAGTCTACGCCCTTG
R: AAAGCCTTTGGCAATGAACA
(tc)7
The average number of alleles per primer pairs was
3.22, ranging from 1 to 6. The PIC values obtained
from the SSR primers ranged from 0.00 to 0.75 with a
mean value of 0.546. The primer CAMS-864 was found
to be the most informative locus for fingerprinting and
differentiating the pepper genotypes (Table 4). The
distribution pattern of alleles per SSR locus for SSR1
across the thirteen pepper varieties is shown in Fig. 3.
The Similarity coefficients based on SSR markers
ranged from 0.117 to 0.9 with mean value of 0.39
(Table 3). The dendrogram grouped the 13 genotypes
into three main clusters (Fig. 4). The first cluster further
divided into two sub clusters. The first sub cluster
contains four genotypes (CA 97, CO2, VSH and K1)
erected fruit position grouped in the first cluster. The
second and third clusters showed to form the largest
clusters containing 84.6% of the population studied.
The second cluster contains four chilli genotypes (CCH,
CO2, K1 and CA110) and two paprika varieties (KT PL
19 and KT PL 18). Five genotypes including one chilli
variety (Kashi Anmol) and four paprika (Arka Abhir,
Byadagi Kaddi, V1 and VSH) types were grouped in
the third cluster. The accession CF1 with erect fruit
position made the fourth cluster.
Nine SSR primer pairs were used to study the
genetic diversity or/and similarity of the thirteen pepper
varieties. Of these primer pairs, 8 (88.88%) revealed
polymorphism in cultivars of the chillies and paprika.
28 Asian J. Agric. Sci., 5(2): 25-31, 2013
generally clustered together and those with different
fruit characteristics are clustered apart.
The cluster analysis based on RAPD revealed that
the thirteen genotypes could be classified into four
major groups (Fig. 2). The first cluster containing CA97
from the Capsicum annuum group with medium and
erect fruit position was distantly separated from other
Capsicum annuum genotypes which are having pendent
fruit position. The second and the third cluster comprise
genotypes of medium to large sized fruits with
declining fruit position. The same pattern of variation
was found in the previous study that the cultivar with
medium and erect fruits formed a more divergent group
than the large fruited cultivar and similarly the medium
and long fruited cultivar were closely related to each
other due to their declining fruit position (Sitthiwong
et al., 2005).
One subgroup from the second cluster comprises
the two paprika varieties KT PL 19 and KT PL18 which
are believed to come from the same geographical place
of origin. Similarly, Arka Abhir and Byadagi Kaddi
coming from the same place (Karnataka) were placed in
the third cluster. This grouping further strengthened by
previous findings (Agrama and Tuinstra, 2003; Gupta
et al., 2008) that DNA based data can be used for
studying the genetic relationship among different
genotype of a species based on place of origin.
Interestingly, putting the variety CO2 and the
accession line CA110 in one subgroup may be
explained by their common prostate growth habit
though they have different fruit characteristics. Finally
the most genetically divergent line CF 1 which belongs
to the Capsicum frutescens species having small and
erect fruit position was grouped in the fourth cluster.
This result is in agreement with the previous works
reported on capsicum species (Rodriguez et al., 1999;
Sitthiwong et al., 2005).
The wide variation in genetic similarity coefficient
among the different genotypes reflected a relatively
high level of polymorphism at the DNA level. The
highest similarity (0.91) was found between KTPL 19
and KTPL 18 (Table 4). The reason was probably due
to that both KTPL 19 and KTPL 18 belong to
C. annuum and originated from the same place.
Rodriguez et al. (1999) and Ince et al. (2009) reported
the large genetic variation presented among different
groups of capsicum species. Likewise, Bydaggi kaddi
and CF1 which belongs to different group of capsicum
species showed the least similarity coefficient (0.36)
displaying wide genetic difference between the
Capsicum annuum and Capsicum frutescens.
SSR markers are considered more reliable because
of their ability to produce highly consistent profiles
(McCouch et al., 1997). The twenty-eight polymorphic
SSR markers clearly differentiated the 13 chilli and
paprika varieties/accession.
Fig. 3: PCR amplification pattern obtained from thirteen
capsicum genotypes using SSR primer CAMS-117
Lane 1: CA97; 2: CCH; 3: CF1; 4: CO2; 5: K1; 6:
CA110; 7: Kashi anmol; 8: Arka abhir; 9: KT PL 19;
10: KT PL 18; 11: Byadagi kaddi; 12: V1; 13: VSH;
M: Molecular size marker (100 bp ladder, Gene link)
Fig. 4: Dendrogram of thirteen pepper accessions and
varieties using UPGMA cluster analysis based on SSR
markers and the second sub cluster contained six genotypes
(Arka Abhir, Byadagi kaddi, Kashi Anmol, V1, CA110
and CCH). The two paprika varieties KTPL19 and
KTPL18 were grouped in the second cluster while only
one cultivar CF1 was found to make the third major
cluster.
DISCUSSION
Understanding genetic variability’s presented
among cultivars or accessions have practical application
in protecting genotypes, conservation and widening the
genetic basis of varieties (Charcosset et al., 1998). The
polymorphism revealed by RAPD primers used in the
study is in consistence with previous reports where an
average of 4.96 and 6.4 polymorphic bands/primer was
detected in pepper genotypes (Rodriguez et al., 1999;
Litoriya et al., 2009). However, the proportion of
polymorphic fragments was comparatively lower than
those earlier reports as observed on an average of 11.2
polymorphic bands per primer using tomato varieties
(Abd El-Hady et al., 2010).
The classification obtained with RAPD markers is
generally consistent with the varietal group, that is,
genotypes with similar fruit and growth characteristics
29 Asian J. Agric. Sci., 5(2): 25-31, 2013
The average numbers of alleles per SSR primer
pairs were in agreement with earlier works reported in
pepper with the mean value of 3.5 alleles/locus
(Hanáček et al., 2009). In addition, the average PIC
values of 0.546 from this study were similar to the PIC
values for pepper (0.529) and Brassica (0.55) (Kwon
et al., 2005; Tonguç and Griffiths, 2004).
Similar to the RAPD clustering pattern the SSR
markers put the accession CF1 in one single major
cluster from the large fruited types of Capsicum
annuum accessions (Fig. 4). These genetic relationships
between the accessions were in good agreement with
the previous reports on pepper genotypes (Portis et al.,
2007). Moreover the two paprika varieties (KT PL19
and KTPL 18) which were grouped together using the
RAPD primer were again clustered in one major cluster
based on the SSR markers. This consistency between
RAPD and SSR in clustering genotypes into the same
group was shown by closer placement of Arka Abhir
and Kashi Anmol in both dendrograms. In contrary, the
cultivar CA97 with medium and erect fruit position was
closely clustered with the variety CO2 with small and
bold type fruit. This may be explained by that mostly
SSR markers measures genetic variation mainly in noncoding sequences which possibly do not have a major
impact on the morphology of genotype (Kwon et al.,
2005).
Interestingly, the clusters were also found to
contain genotypes from both the pungent (Chilli) and
the non pungent (Paprika) types. This indicates that
capsaicinoid profiles are not good taxonomic indicators
for the capsicum species (Zewdie and Bosland, 2001).
The pairwise similarity analysis of the SSR
markers produced an index values ranging from 0.117
to 0.9 with a lesser mean value of 0.39 when compared
to RAPD (0.6) mean value. Moreover, most of the
similarity index values from the SSR analysis stretched
out in the range between 0.117 and 0.6 when compared
to RAPD analysis where most of the similarity index
values found between 0.4 and 0.85. This indicates that
SSR markers have a different polymorphic capability
which resulted in wider genetic associations than
displayed by the RAPD markers (Powell et al., 1996).
ACKNOWLEDGMENT
This work was supported in part by Cadbury and
NAIP schemes, Tamil Nadu agricultural university,
India.
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